A single nucleotide polymorphism (SNP) is a single base variation in the genome of a living organism. SNPs may occur in coding sequences of genes and non-coding regions of genes, including regulatory regions. SNPs in the coding sequences of the genome are classified as two types: synonymous and nonsynonymous. Synonymous SNPs do not alter the protein sequence due to the degeneracy of the genetic code, while nonsynonymous SNPs change the amino acid sequence of the encoded protein. The nonsynonymous SNPs are further divided into two types: missense and nonsense. A missense mutation is a single nucleotide point mutation leading to a codon that codes for a different amino acid compared to the wild-type, whereas a nonsense mutation is a point mutation that results in a premature stop codon. SNPs that are not in protein-coding regions can impact the function of the genes by altering splicing sequences and binding activity of transcription factors as well as gene expression. Among all the genetic variations, SNPs are the most common genetic differences between human beings. Over 3.1 million SNPs have been characterized from the human genome in a second-generation human haplotype map (Frazer et al. 2007). Thus, SNPs are important biomarkers for investigating the molecular basis underlying the mechanism for disease development, laying a foundation for precision medicine.
The Human Genome Project and the construction of a comprehensive human genome sequence map (Lander et al. 2001, Venter et al. 2001, and Wheeler et al. 2008) provide valuable resources for the study of genetic variations. These genetic differences include SNPs, gene copy number variations, insertions and deletions. SNPs have been established as unique biomarkers for the discovery and characterization disease genes (Kwok 2000 and Roses 2000). These research efforts require the characterization of large number of SNPs with technologies that are cost-effective and high-throughput with high-accuracy. The following DNA sequencing platforms are widely used for characterizing genetic variations: (1) 4-color fluorescent Sanger method (Smith et al. 1986, Ju et al. 1995, Ju et al. 1996, Salas-Solano et al. 1998, and Kheterpal et al. 1996), (2) sequencing by synthesis (SBS) using cleavable fluorescent nucleotide reversible terminators (Ju et al. 2006 and Bentley et al. 2008), (3) SBS with detection of the chemiluminescent signals caused by the released pyrophosphate during polymerase reaction (pyrosequencing) (Margulies et al. 2005), (4) SBS with electronic detection of the released proton during polymerase reaction (ion torrent sequencing) (Rothberg et al. 2011), and (5) single molecule fluorescent SBS methods (Harris et al. 2008 and Eid et al. 2009). However, these sequencing technologies are not designed for pinpoint detection of SNPs, and are still too costly for performing large scale SNP studies. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence emission are two dominant detection methods for SNP analysis. SNP assay approaches using the above two detection methods are reviewed below.
SNP Analysis by MALDI-TOF MS Detection
MALDI-TOF MS measures the mass of the target molecules with highly accurate results in a digital format. It has been used for SNP detection by single base extension (SBE) (Haff et al. 1997, Tang et al. 1999, Ross et al. 1998, Fei et al. 1998, and Griffin et al. 2000), hybridization (Stoerker et al. 2000 and Ross et al. 1997), and invasive cleavage (Griffin et al. 1999 and Lyamichev et al. 1999). MALDI-TOF MS has also been used for gene expression analysis and single-copy DNA haplotyping in the context of nucleotide extension by polymerase (Ding et al., PNAS 100:3059-3064, 2003 and Ding et al., PNAS 100:7449-7453 2003).
Most multiplex SNP analyses make use of the specificity of the SBE reaction catalyzed by polymerase. One of the widely used SNP characterization method utilizes SBE and MALDI-TOF MS detection. In this approach, oligonucleotide primers are designed and synthesized based on the genetic variation in the target gene. The 3′-end of the primer anneals immediately next to a SNP site of the DNA template. A single dideoxynucleotide that is complementary to the SNP site is then incorporated into the primer by DNA polymerase. The identity of the SNP is determined by the mass of the resulting primer extension product obtained from the MALDI-TOF MS spectrum.
SNP Analysis by Fluorescence Detection
Numerous SNP genotyping methods have been developed using fluorescence labeling and detection, including microarray (Hartmann et al. 2009), PCR-RFLP analysis (Chowdhury et al. 2007), and TagMan real-time genotyping (Bai et al. 2004). There are several advantages to using fluorescence labeling and detection, which include a variety of robust chemical coupling methods to tag the target molecules, high detection sensitivity of several photophysical parameters (life time, emission and polarization) and the capability of multiplexing. The molecular inversion probe (MIP) approach has been developed for SNP detection (Hardenbol et al. 2003). In this method, successive extension and ligation of locus-specific DNA probes yields a circular shape at polymorphic sites of the target gene. The linear probes are then selectively degraded, whereas the circular DNA probes that contain allelic information are amplified and analyzed using a microarray with fluorescence detection. Using this approach, Hardenbol et al. (2003) performed genotyping of more than 1,000 SNPs per assay. The HIP method has the advantage of a very high level of multiplexing. However, many enzymatic reaction steps and complicated probe design are required for NIP.
Prior multiplex SNP assays primarily used either mass spectrometric detection or fluorescent tags and optical detection. None of these previous assays offer single molecule detection sensitivity and all require bulky instruments. None used nanopores to identify molecular or polymer tags corresponding to nucleotides of interest or SNPs, so as to identify the nucleotides of interest or SNPs.